Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain

The activity of calmodulin-like domain protein kinase (CDPK) is regulated by the direct binding of Ca2+. Unmodified soybean CDPK alpha and a chimeric enzyme in which the calmodulin-like domain (CLD) was replaced by VU-1 calmodulin had similar values of V-maxapp (3.19, 3.46, and 3.60, 3.93 mu mol/min/mg, respectively), and each was activated 30-70-fold by Ca2+ To determine if activation results from the binding of the CLD to the autoinhibitory (junction) domain of CDPK alpha in a manner analogous to the activation of calmodulin-dependent enzymes by calmodulin, recombinant CLD and truncation mutants of CDPK alpha were expressed in bacteria and highly purified. In blot overlays, biotinylated CLD bound to mutants containing residues 312-328 of the junction domain. In an electrophoretic mobility shift assay CLD bound synthetic peptides containing residues 318-332 in a calcium-dependent manner, providing direct evidence for binding of CLD to a site in the junction domain. Mutants of CDPK alpha from which all or part of the CLD had been deleted were constitutively inactive. Addition of 20 mu M CLD to these mutants in the presence, but not the absence, of calcium stimulated their activities, but to various degrees, His(6)-CDPK alpha(1-328), which contained none of the CLD, was activated only 5-fold, but the activity of His6-CDPK alpha(1-398), which retained nearly half of the CLD in its sequence, was stimulated 64-fold. The latter activity approached that of unmodified CDPK alpha and was half maximal at a CLD concentration of 7 mu M. Our results suggest that binding of CLD to the junction domain contributes to, but is not sufficient for activation. Although calmodulin supported full activity of the chimeric enzyme, its addition to His(6)-CDPK alpha(1-398) resulted in activity that was only 6% of that of the unmodified enzyme and which was half-maximal at 20 mu M Arabidopsis calmodulin. These results support the conclusion that simple binding of the calmodulin-like domain to the junction domain is not sufficient for activation.